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Human monoclonal antibody specifically binding to envelope protein Gn of severe fever with thrombocytopenia syndrome virus and application thereof

A technology for cloning antibodies and platelets, applied in the field of medicine, can solve problems such as no specific drugs

Active Publication Date: 2022-07-12
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no clinically available specific drugs

Method used

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  • Human monoclonal antibody specifically binding to envelope protein Gn of severe fever with thrombocytopenia syndrome virus and application thereof
  • Human monoclonal antibody specifically binding to envelope protein Gn of severe fever with thrombocytopenia syndrome virus and application thereof
  • Human monoclonal antibody specifically binding to envelope protein Gn of severe fever with thrombocytopenia syndrome virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Expression and purification of the extracellular segment of SFTSV Gn

[0048]The coding sequence of 6 histidine tags (6×histag) and translation stop codons were connected to the 3' end of the coding region of the extracellular segment of the SFTSV Gn protein (SEQ ID NO: 9), and constructed into the pFastBac1 vector by EcoRI and XhoI (purchased from Invitrogen). The ligation product was then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. The recombinant baculovirus was extracted, transfected into sf9 cells (purchased from Invitrogen) for baculovirus packaging, and then amplified by the virus, added to Hi5 cells (purchased from Invitrogen) for SFTSV Gn extracellular segment protein expression.

[0049] After the cell culture medium containing the target protein is purified by nickel ion affinity chromatography (HisTrapTMHP(GE)) and gel filtration chromatography (SuperoseTM6Increase 10 / 300GL(GE)), a relatively ...

Embodiment 2

[0050] Example 2: Isolation of SFTSV Gn protein-specific memory B cells

[0051] 15mL of blood was collected to isolate PBMCs with the informed consent of a person who was cured and discharged after SFTSV infection (1 person from Beijing Ditan Hospital Affiliated to Capital Medical University). The isolated PBMCs were divided into 10 7 The density of / mL was combined with the purified SFTSV Gn extracellular segment protein in Example 1 with a final concentration of 400nM, which was incubated on ice for half an hour, then washed twice with PBS, and then mixed with the following antibodies (all purchased from BD, using both 10μg / mL) were incubated with: anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After antibody incubation on ice for half an hour, PBMCs were washed twice with PBS.

[0052] PBS-washed PBMCs were sorted by FACSAria III to...

Embodiment 3

[0053] Example 3: Single B cell PCR, sequence analysis and human antibody design

[0054] According to the method described in Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus published by Qihui Wang et al. in Science Translational Medicine, Vol. 8, No. 369 in December 2016, the B cells obtained in Example 2 were treated with Reverse transcription was performed by Superscript III reverse transcriptase (Invitrogen), and the reverse transcription primers were as shown in Table 1, and the reaction was performed at 55°C for 60 minutes.

[0055] Table 1. Primers for reverse transcription reactions

[0056]

[0057] Using this reverse transcription product as a template, PCR was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). The corresponding primers were designed, and the reaction conditions were as follows: 95°C, 5 min; 95°C for 30s, 55°C (heavy chain / κ chain) for ...

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Abstract

The invention relates to a human monoclonal antibody specifically combined with envelope protein Gn of severe fever with thrombocytopenia syndrome virus and application of the human monoclonal antibody. The antibody can specifically treat infection of severe fever with thrombocytopenia syndrome virus.

Description

technical field [0001] The invention belongs to the technical field of medicine, and particularly relates to a human monoclonal antibody that specifically binds to the envelope protein Gn of fever with thrombocytopenia syndrome virus and uses thereof. Background technique [0002] In 2009, the fatal incident of tick bites occurred in Henan and other places in China. Scientists identified that it was caused by a new type of Bunia virus (Fever with Thrombocytopenia Syndrome Virus, SFTSV). The virus is transmitted through tick bites, and cases have been reported in more than 20 provinces and cities in my country, as well as in South Korea, Japan, and Vietnam. Infected patients develop acute fever, accompanied by thrombocytopenia and leukopenia, gastrointestinal dysfunction and other symptoms. Severe patients will develop multiple organ failure or even die, with a mortality rate of 12-30%. The World Health Organization has listed SFTSV as one of the viruses of greatest concern....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/395A61P31/14
CPCC07K16/10A61P31/14C07K2317/565A61K2039/505Y02A50/30
Inventor 吴燕高峰李世华高福
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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